Astrocytic Regulation of Cocaine Locomotor Sensitization in EcoHIV Infected Mice.

Cocaine use disorder (CUD) is highly comorbid with HIV infection and worsens HIV outcomes. Preclinical research on the outcomes of HIV infection may yield crucial information on neurobehavioral changes resulting from chronic drug exposure in people living with HIV (PLWH). Repeated exposure to cocaine alters behavioral responses to cocaine. This includes development of cocaine locomotor sensitization - or increased locomotor responses to the same doses of cocaine - which depends on nucleus accumbens (NAc) neural plasticity. NAc astrocytes are key regulators of neural activity and plasticity, and their function can be impaired by cocaine exposure and HIV infection, thus implicating them as potential regulators of HIV-induced changes in behavioral response to cocaine. To characterize the effects of HIV infection on cocaine locomotor sensitization, we employed the EcoHIV mouse model to assess changes in locomotor responses after repeated cocaine (10mg/kg) exposure and challenge. EcoHIV infection potentiated expression of cocaine sensitization. We also identified EcoHIV-induced increases in expression of the astrocytic nuclear marker Sox9 selectively in the NAc core. To investigate whether modulation of NAc astrocytes could reverse EcoHIV-induced deficits, we employed a chemogenetic approach. We found that chemogenetic activation of NAc astrocyte Gq signaling attenuated EcoHIV-enhanced cocaine sensitization. We propose that HIV infection contributes to cocaine behavioral sensitization and induces adaptations in NAc astrocytes, while promoting NAc astrocytic Gq-signaling can recover EcoHIV-induced behavioral changes. These findings identify potential cellular substrates of disordered cocaine-driven behavior in the context of HIV infection and point toward strategies to reduce cocaine-related behavior in PLWH.

Effects of Antiretroviral Treatment on Central and Peripheral Immune Response in Mice with EcoHIV Infection.

HIV infection is an ongoing global health issue, despite increased access to antiretroviral therapy (ART). People living with HIV (PLWH) who are virally suppressed through ART still experience negative health outcomes, including neurocognitive impairment. It is increasingly evident that ART may act independently or in combination with HIV infection to alter the immune state, though this is difficult to disentangle in the clinical population. Thus, these experiments used multiplexed chemokine/cytokine arrays to assess peripheral (plasma) and brain (nucleus accumbens; NAc) expression of immune targets in the presence and absence of ART treatment in the EcoHIV mouse model. The findings identify the effects of EcoHIV infection and of treatment with bictegravir (B), emtricitabine (F), and tenofovir alafenamide (TAF) on the expression of numerous immune targets. In the NAc, this included EcoHIV-induced increases in IL-1α and IL-13 expression and B/F/TAF-induced reductions in KC/CXCL1. In the periphery, EcoHIV suppressed IL-6 and LIF expression, while B/F/TAF reduced IL-12p40 expression. In the absence of ART, IBA-1 expression was negatively correlated with CX3CL1 expression in the NAc of EcoHIV-infected mice. These findings identify distinct effects of ART and EcoHIV infection on peripheral and central immune factors and emphasize the need to consider ART effects on neural and immune outcomes.

Autologous neutralizing antibody responses after antiretroviral therapy in acute and early HIV-1.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after acquisition) and early HIV (60-128 days after acquisition).RESULTSPlasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 envelopes representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In 2 of the 3 acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter.CONCLUSIONResults indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants.TRIAL REGISTRATIONClinicalTrials.gov NCT02656511.FUNDINGNIH grants U01AI169767, R01AI162646, UM1AI164570, UM1AI164560, U19AI096109, K23GM112526, T32AI118684, P30AI045008, P30AI027763, R24AI067039; Gilead Sciences grant INUS2361354; Viiv Healthcare grant A126326.

Effects of antiretroviral treatment on central and peripheral immune response in mice with EcoHIV infection.

HIV infection is an ongoing global health issue despite increased access to antiretroviral therapy (ART). People living with HIV (PLWH) who are virally suppressed through ART still experience negative health outcomes, including neurocognitive impairment. It is increasingly evident that ART may act independently or in combination with HIV infection to alter immune state, though this is difficult to disentangle in the clinical population. Thus, these experiments used multiplexed chemokine/cytokine arrays to assess peripheral (plasma) and brain (nucleus accumbens; NAc) expression of immune targets in the presence and absence of ART treatment in the EcoHIV mouse model. The findings identify effects of EcoHIV infection and of treatment with bictegravir (B), emtricitabine (F) and tenofovir alafenamide (TAF) on expression of numerous immune targets. In the NAc, this included EcoHIV-induced increases in IL-1α and IL-13 expression and B/F/TAF-induced reductions in KC/CXCL1. In the periphery, EcoHIV suppressed IL-6 and LIF expression, while B/F/TAF reduced IL-12p40 expression. In absence of ART, IBA-1 expression was negatively correlated with CX3CL1 expression in the NAc of EcoHIV-infected mice. These findings identify distinct effects of ART and EcoHIV infection on peripheral and central immune factors and emphasize the need to consider ART effects on neural and immune outcomes.

EcoHIV Infection Modulates the Effects of Cocaine Exposure Pattern and Abstinence on Cocaine Seeking and Neuroimmune Protein Expression in Male Mice.

Cocaine use disorders (CUDs) and human immunodeficiency virus (HIV) remain persistent public health dilemmas throughout the world. One major hurdle for treating CUD is the increase in cocaine craving and seeking behavior that occurs over a protracted period of abstinence, an effect known as the incubation of craving. Little is known about how HIV may modulate this process. Thus, we sought to examine the impact of chronic HIV infection on the incubation of cocaine craving and associated changes in the central and peripheral immune systems. Here, mice were inoculated with EcoHIV, which is a chimeric HIV-1 construct that produces chronic HIV infection in mice. EcoHIV- and sham-infected mice were conditioned with cocaine daily or intermittently in a conditioned place preference (CPP) paradigm, followed by 1 or 21 days of forced abstinence prior to assessing preference for the cocaine-paired chamber. Under both conditioning regimens, sham mice exhibited incubation of cocaine CPP after 21 days of abstinence. EcoHIV-infected mice conditioned daily with cocaine showed enhanced cocaine seeking at both abstinence timepoints, whereas infected mice conditioned intermittently showed a reversal of the incubation effect, with higher cocaine seeking after 1 day of abstinence compared to 21 days. Analysis of corticolimbic CX3CL1-CX3CR1 and glutamate receptor expression revealed alterations in medial prefrontal cortex (mPFC) CX3CL1 and nucleus accumbens (NAc) GluN2A receptors that correlated with cocaine seeking following daily cocaine exposure. Moreover, examination of peripheral immune markers showed that the effect of abstinence and EcoHIV infection on these measures depended on the cocaine exposure regimen. Altogether, these results highlight the importance of cocaine abstinence and exposure pattern as critical variables that modulate HIV-associated neuroimmune outcomes and relapse vulnerability.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between HIV infection and cocaine use disorder is likely bidirectional, with cocaine use directly impacting immune function while HIV infection alters addiction-related behavior. To better characterize the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilizes a humanized mouse model to investigate the outcomes of HIV-1 infection on cocaine-related behaviors in a conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection selectively impairs cocaine CPP extinction without effecting reinstatement or cocaine seeking under conflict. Behavioral alterations are accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes are observed in human cytokines, including HIV-induced reductions in human TNFα, and cocaine and HIV interactions on GM-CSF levels. Together these data provide new insights into the unique neurobehavioral outcomes of HIV infection and cocaine exposure and how they interact to effect immune responses.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between HIV infection and cocaine use disorder is likely bidirectional, with cocaine use directly impacting immune function while HIV infection alters addiction-related behavior. To better characterize the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilized a humanized mouse model to investigate the outcomes of HIV-1 infection on cocaine-related behaviors in a conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection selectively impaired cocaine CPP extinction without effecting reinstatement or cocaine seeking under conflict were observed. Behavioral alterations were accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes were observed in both mouse and human cytokines, including HIV-induced reductions in mouse IL-1α and G-CSF and human TNFα, and cocaine induced alterations in mouse GM-CSF. Together these data provide new insights into the unique neurobehavioral outcomes of HIV infection and cocaine exposure and how they interact to effect immune responses.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between progressive HIV infection and cocaine use disorder is likely bidirectional, with cocaine use having direct effects on immune function while HIV infection can alter addiction-related behavior. To better characterized the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilized a humanized mouse model to investigate the outcomes of progressive HIV infection on cocaine-related behaviors in a cocaine conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection did not impact the formation of a cocaine CPP, but did result in resistance to extinction of the CPP. No effects of HIV on yohimbine-primed reinstatement or cocaine seeking under conflict were observed. These behavioral alterations were accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes were observed in both mouse and human markers. Among other targets, this included HIV-induced reductions in mouse IL-1α and G-CSF and human TNFα and cocaine-induced alterations in human TNFα and mouse GM-CSF such that cocaine exposure increases both cytokines only in the absence of HIV infection. Together these data provide new insights into the unique neurobehavioral processes underlying HIV infection and cocaine use disorders, and further how they interact to effect immune responses.

Fire Properties of Bed Mattresses Focusing on the Fire Growth Rate and Flame Height.

Bed mattresses are rated as products to cause a fire hazard because of their very high heat release rate among indoor combustibles. In this study, fire growth rate and flame height were measured through a series of combustion experiments on a full scale in order to provide information regarding mattress fire characteristics. The experiments were conducted in an open space, and bed mattresses as the test samples were installed at different installation heights (0~515 mm). The experiment results revealed that the higher the bed mattress was installed, the higher the fire growth rate, the heat release rate, and the flame height. Additionally, the time of the mattress to reach 1 MW was evaluated as the category "medium" in the NFPA 72 standards. The flame heights showed a good coincidence compared to the existing flame height model equations, proving the applicability of the model to the mattress combustion.

Late Inflammation Induced by Asbestiform Fibers in Mice Is Ameliorated by a Small Molecule Synthetic Lignan.

Exposure to Libby amphibole (LA) asbestos-like fibers is associated with increased risk of asbestosis, mesothelioma, pulmonary disease, and systemic autoimmune disease. LGM2605 is a small molecule antioxidant and free radical scavenger, with anti-inflammatory effects in various disease models. The current study aimed to determine whether the protective effects of LGM2605 persist during the late inflammatory phase post-LA exposure. Male and female C57BL/6 mice were administered daily LGM2605 (100 mg/kg) via gel cups for 3 days before and 14 days after a 200 µg LA given via intraperitoneal (i.p.) injection. Control mice were given unsupplemented gel cups and an equivalent dose of i.p. saline. On day 14 post-LA treatment, peritoneal lavage was assessed for immune cell influx, cytokine concentrations, oxidative stress biomarkers, and immunoglobulins. During the late inflammatory phase post-LA exposure, we noted an alteration in trafficking of both innate and adaptive immune cells, increased pro-inflammatory cytokine concentrations, induction of immunoglobulin isotype switching, and increased oxidized guanine species. LGM2605 countered these changes similarly among male and female mice, ameliorating late inflammation and altering immune responses in late post-LA exposure. These data support possible efficacy of LGM2605 in the prolonged treatment of LA-associated disease and other inflammatory conditions.

Copper Oxide Nanoparticle-Induced Acute Inflammatory Response and Injury in Murine Lung Is Ameliorated by Synthetic Secoisolariciresinol Diglucoside (LGM2605).

Metal-oxide nanoparticles (MO-NPs), such as the highly bioreactive copper-based nanoparticles (CuO-NPs), are widely used in manufacturing of hundreds of commercial products. Epidemiological studies correlated levels of nanoparticles in ambient air with a significant increase in lung disease. CuO-NPs, specifically, were among the most potent in a set of metal-oxides and carbons studied in parallel regarding DNA damage and cytotoxicity. Despite advances in nanotoxicology research and the characterization of their toxicity, the exact mechanism(s) of toxicity are yet to be defined. We identified chlorination toxicity as a damaging consequence of inflammation and myeloperoxidase (MPO) activation, resulting in macromolecular damage and cell damage/death. We hypothesized that the inhalation of CuO-NPs elicits an inflammatory response resulting in chlorination damage in cells and lung tissues. We further tested the protective action of LGM2605, a synthetic small molecule with known scavenging properties for reactive oxygen species (ROS), but most importantly, for active chlorine species (ACS) and an inhibitor of MPO. CuO-NPs (15 µg/bolus) were instilled intranasally in mice and the kinetics of the inflammatory response in lungs was evaluated 1, 3, and 7 days later. Evaluation of the protective action of LGM2605 was performed at 24 h post-challenge, which was selected as the peak acute inflammatory response to CuO-NP. LGM2605 was given daily via gavage to mice starting 2 days prior to the time of the insult (100 mg/kg). CuO-NPs induced a significant inflammatory influx, inflammasome-relevant cytokine release, and chlorination damage in mouse lungs, which was mitigated by the action of LGM2605. Preventive action of LGM2605 ameliorated the adverse effects of CuO-NP in lung.

Assessment of a Small Molecule Synthetic Lignan in Enhancing Oxidative Balance and Decreasing Lipid Accumulation in Human Retinal Pigment Epithelia.

Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 ß release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 ß release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits Libby amphibole fiber-induced acute inflammation in mice.

BACKGROUND: Exposure to the Libby amphibole (LA) asbestos-like fibers found in Libby, Montana, is associated with inflammatory responses in mice and humans, and an increased risk of developing mesothelioma, asbestosis, pleural disease, and systemic autoimmune disease. Flaxseed-derived secoisolariciresinol diglucoside (SDG) has anti-inflammatory, anti-fibrotic, and antioxidant properties. We have previously identified potent protective properties of SDG against crocidolite asbestos exposure modeled in mice. The current studies aimed to extend those findings by evaluating the immunomodulatory effects of synthetic SDG (LGM2605) on LA-exposed mice. METHODS: Male and female C57BL/6 mice were given LGM2605 via gavage initiated 3 days prior to and continued for 3 days after a single intraperitoneal dose of LA fibers (200 µg) and evaluated on day 3 for inflammatory cell influx in the peritoneal cavity using flow cytometry. RESULTS: LA exposure induced a significant increase (p < 0.0001) in spleen weight and peritoneal influx of white blood cells, all of which were reduced with LGM2605 with similar trends among males and females. Levels of peritoneal PMN cells were significantly (p < 0.0001) elevated post LA exposure, and were significantly (p < 0.0001) blunted by LGM2605. Importantly, LGM2605 significantly ameliorated the LA-induced mobilization of peritoneal B1a B cells. CONCLUSIONS: LGM2605 reduced LA-induced acute inflammation and WBC trafficking supporting its possible use in mitigating downstream LA fiber-associated diseases. SUMMARY: Following acute exposure to Libby amphibole (LA) asbestos-like fibers, synthetic SDG (LGM2605), a small synthetic molecule, significantly reduced the LA-induced increase in spleen weight and peritoneal inflammation in C57BL/6 male and female mice. Our findings highlight that LGM2605 has immunomodulatory properties and may, thus, likely be a chemopreventive agent for LA-induced diseases.

LGM2605 Reduces Space Radiation-Induced NLRP3 Inflammasome Activation and Damage in In Vitro Lung Vascular Networks.

Updated measurements of charged particle fluxes during the transit from Earth to Mars as well as on site measurements by Curiosity of Martian surface radiation fluxes identified potential health hazards associated with radiation exposure for human space missions. Designing mitigation strategies of radiation risks to astronauts is critical. We investigated radiation-induced endothelial cell damage and its mitigation by LGM2605, a radioprotector with antioxidant and free radical scavenging properties. We used an in vitro model of lung vascular networks (flow-adapted endothelial cells; FAECs), exposed to gamma rays, low/higher linear energy transfer (LET) protons (3⁻4 or 8⁻10 keV/µm, respectively), and mixed field radiation sources (gamma and protons), given at mission-relevant doses (0.25 gray (Gy)⁻1 Gy). We evaluated endothelial inflammatory phenotype, NLRP3 inflammasome activation, and oxidative cell injury. LGM2605 (100 µM) was added 30 min post radiation exposure and gene expression changes evaluated 24 h later. Radiation induced a robust increase in mRNA levels of antioxidant enzymes post 0.25 Gy and 0.5 Gy gamma radiation, which was significantly decreased by LGM2605. Intercellular cell adhesion molecule-1 (ICAM-1) and NOD-like receptor protein 3 (NLRP3) induction by individual or mixed-field exposures were also significantly blunted by LGM2605. We conclude that LGM2605 is a likely candidate to reduce tissue damage from space-relevant radiation exposure.

Blockade of Tumor-Expressed PD-1 promotes lung cancer growth.

Anti-PD-1 immunotherapy is the standard of care for treating many patients with non-small cell lung cancer (NSCLC), yet mechanisms of treatment failure are emerging. We present a case of NSCLC, who rapidly progressed during a trial (NCT02318771) combining palliative radiotherapy and pembrolizumab. Planned tumor biopsy demonstrated PD-1 expression by NSCLC cells. We validated this observation by detecting PD-1 transcript in lung cancer cells and by co-localizing PD-1 and lung cancer-specific markers in resected lung cancer tissues. We further investigated the biological role of cancer-intrinsic PD-1 in a mouse lung cancer cell line, M109. Knockout or antibody blockade of PD-1 enhanced M109 viability in-vitro, while PD-1 overexpression and exposure to recombinant PD-L1 diminished viability. PD-1 blockade accelerated growth of M109-xenograft tumors with increased proliferation and decreased apoptosis in immune-deficient mice. This represents a first-time report of NSCLC-intrinsic PD-1 expression and a potential mechanism by which PD-1 blockade may promote cancer growth.

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605) Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner.

Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2) activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT) and Nrf2 disrupted (Nrf2-/-) mice. Cells were pretreated with LGM2605 (50 µM and 100 µM) and exposed to asbestos fibers (20 µg/cm²) and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1ß (IL-1ß), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα)), cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2-/- macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation.

PD-1 Modulates Radiation-Induced Cardiac Toxicity through Cytotoxic T Lymphocytes.

INTRODUCTION: Combined immune checkpoint blockade has led to rare autoimmune complications, such as fatal myocarditis. Recent approvals of several anti-programmed death 1 (anti-PD-1) drugs for lung cancer treatment prompted ongoing clinical trials that directly combine PD-1 inhibitors with thoracic radiotherapy for locally advanced lung cancer. Overlapping toxicities from either modality have the potential to increase the risk for radiation-induced cardiotoxicity (RICT), which is well documented among patients with Hodgkin's disease and breast cancer. METHODS: To investigate cardiotoxicity without the compounding pulmonary toxicity from thoracic radiotherapy, we developed a technique to deliver cardiac irradiation (CIR) in a mouse model concurrently with PD-1 blockade to determine the presence of cardiac toxicity by using physiological testing and mortality as end points along with histological analysis. RESULTS: We observed an acute mortality of 30% within 2 weeks after CIR plus anti-PD-1 antibody compared with 0% from CIR plus immunoglobulin G (p = 0.023). Physiological testing demonstrated a reduced left ventricular ejection fraction (p < 0.01) by echocardiogram. Tissue analyses revealed increased immune cell infiltrates within cardiac tissue. Depletion of CD8-positive lymphocytes with anti-CD8 antibody reversed the acute mortality, suggesting that the toxicity is CD8-positive cell-mediated. To validate these findings using a clinically relevant fractionated radiotherapy regimen, we repeated the study by delivering five daily fractions of 6 Gy. Similar mortality, cardiac dysfunction, and histological changes were observed in mice receiving fractionated radiotherapy with concurrent anti-PD-1 therapy. CONCLUSIONS: This study provides strong preclinical evidence that radiation-induced cardiotoxicity is modulated by the PD-1 axis and that PD-1 blockade should be administered with careful radiotherapy planning with an effort of reducing cardiac dose.

Familial and Somatic BAP1 Mutations Inactivate ASXL1/2-Mediated Allosteric Regulation of BAP1 Deubiquitinase by Targeting Multiple Independent Domains.

Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and in vitro reconstitution biochemistry. We found that the BAP1-ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable.Significance: Combined computational and biochemical approaches demonstrate that the BAP1-ASXL2 interaction is direct and high affinity and that many BAP1 mutations act allosterically to inhibit BAP1-ASXL2 binding. Cancer Res; 78(5); 1200-13. ©2017 AACR.

Hydrolysis of Hyaluronic Acid in Lymphedematous Tissue Alleviates Fibrogenesis via TH1 Cell-Mediated Cytokine Expression.

Although surgery and radiation are beneficial for treating cancer, they can also lead to malfunctions of the lymphatic system such as secondary lymphedema. This abnormality of the lymphatic system is characterized by severe swelling, adipogenesis, inflammation, and fibrosis in the lymphedematous region. Moreover, the proliferation of fibrotic tissue in the lymphedematous region generates edema that is no longer spontaneously reversible. No treatment for fibrosis has been validated in patients with lymphedema. In our efforts to develop a therapeutic agent for lymphedema fibrosis, we used a newly established mouse hind limb model. Previous studies have demonstrated that hyaluronic acid accumulates in the lymphedematous region. Thus, we challenged mice with of hyaluronidase (HYAL), with the aim of reducing fibrogenesis. After subcutaneous injections in the lymphedematous mouse leg every two days, the volume of lymphedema had reduced significantly by 7 days post-operation. Histochemical analysis indicated that collagen accumulation and myofibroblast differentiation were decreased in epidermal tissues after HYAL injection. Moreover, it was associated with upregulation of interferon-gamma, increased numbers of Th1 cells, and downregulation of interleukin-4 and interleukin-6 in the lymphedematous region and spleen. These results indicate that hydrolysis of hyaluronic acid can boost an anti-fibrotic immune response in the mouse lymphedema model.

Therapeutic effects of hyaluronidase on acquired lymphedema using a newly developed mouse limb model.

Acquired lymphedema is one of the most dreaded side effects of cancer treatment, such as surgical treatment or irradiation. However, due to the lack of appropriate animal models, there is no effective therapeutic method to cure acquired lymphedema. To develop a reproducible acquired lymphedema animal model, we devised a mouse hind limb model by removing a superficial inguinal lymph node, a popliteal lymph node, a deep inguinal lymph node, and the femoral lymphatic vessel. We measured the volume of lymphedematous leg and observed the change in level of hyaluronic acid (HA) and lymphangiogenic factors after injecting hyaluronidase. Our model showed the distinguishable swelling and the reliable symptoms compared to previously reported models. In the lymphedematous regions of our model, we confirmed that HA, a major component of extracellular matrix, accumulated to higher levels than in a normal mouse. This lymphedema volume was rapidly reduced by treating hyaluronidase. Following hyaluronidase injection, the lymphedematous region of our model resembled a normal hind limb. Our findings indicated that hyaluronidase promoted lymphangiogenesis on the lymphedematous limb. Based on hyaluronidase treatment in the lymphedematous region, this could potentially be a new therapeutic approach for acquired lymphedema mediated through the modification of the size of HA fragments. Impact statement In this manuscript, the essence of the work described in this manuscript involves the development of (1) a mouse limb model showing acquired lymphedema and (2) a potent therapeutic treatment using hyaluronidase to remedy acquired lymphedema in our model. In order to develop a reproducible acquired lymphedema animal model that reflects the most common symptoms experienced by lymphedema patients, we devised a mouse hind limb model by removing lymph nodes and lymphatics. Our model showed the distinguishable swelling and the reliable symptoms compared to previously reported models. In the lymphedematous regions of our model, we confirmed that hyaluronic acid (HA) accumulated to higher levels than in a normal mouse. This lymphedema volume was rapidly reduced by treating the lymphedematous leg with hyaluronidase, which also degraded high molecular weight HA to low molecular weight HA. Immunohistochemical analysis, quantitative real-time PCR analysis and lymphangioscintigraphy showed that hyaluronidase enhanced lymphangiogenesis in the lymphedematous limb.